Compositions and methods for nucleic acid processing using blocking agents

Abstract

Methods and systems for sample preparation techniques that prevent inhibition of reactions such as due to the presence of longer oligonucleotides are presented herein. The methods and systems provided herein allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells.

1 .- 61 . (canceled) 
     
     
         62 . A method, comprising:
 (a) providing a reaction mixture comprising:
 (i) an enzyme; 
 (ii) a first nucleic acid molecule; and 
 (iii) a second nucleic acid molecule comprising a first region and a second region, wherein: (A) the first region is associated with a blocking agent; (B) when the enzyme is bound to the first region in absence of the blocking agent, activity of the enzyme is inhibited; and (C) the second region is disposed at or near a 3′ end of the nucleic acid molecule; and 
   (b) using the enzyme, the first nucleic acid molecule, and the second region of the second nucleic acid molecule to generate a third nucleic acid molecule.   
     
     
         63 . The method of  claim 62 , further comprising partitioning the reaction mixture into a partition, wherein the partition is selected from the group consisting of an aqueous droplet in an emulsion, a well, and an aqueous droplet in an emulsion trapped in a well. 
     
     
         64 . The method of  claim 62 , wherein the second nucleic acid molecule further comprises an azide modified nucleotide, and wherein the blocking agent is an azide-binding blocking agent. 
     
     
         65 . The method of  claim 62 , wherein the second nucleic acid molecule further comprises a biotinylated nucleotide, and wherein the blocking agent is the biotinylated nucleotide or a biotin-binding agent. 
     
     
         66 . The method of  claim 62 , wherein the second nucleic acid molecule further comprises a sequence capable of recognition by a nuclease, and wherein the blocking agent is an inactivated nuclease. 
     
     
         67 . The method of  claim 66 , wherein the inactivated nuclease is a restriction endonuclease. 
     
     
         68 . The method of  claim 62 , wherein the blocking agent is a DNA binding protein or a DNA binding domain of a DNA binding protein. 
     
     
         69 . The method of  claim 68 , wherein the DNA binding protein is a transcription activator-like (TAL) effector or zinc finger protein. 
     
     
         70 . The method of  claim 62 , further comprising removing or releasing the blocking agent from the second nucleic acid molecule. 
     
     
         71 . The method of  claim 70 , wherein the blocking agent is removed or released from the second nucleic acid molecule subsequent to (b). 
     
     
         72 . The method of  claim 62 , wherein the second nucleic acid molecule further comprises one or more additional regions selected from the group consisting of an adapter sequence, a primer or primer binding sequence, a barcode sequence, a unique molecular index (UMI), and a sequence configured to attach to a flow cell of a sequencer. 
     
     
         73 . The method of  claim 72 , wherein the primer or primer binding sequence is a sequencing primer or sequencing primer binding sequence. 
     
     
         74 . The method of  claim 62 , wherein the second region is capable of hybridizing to the first nucleic acid molecule. 
     
     
         75 . The method of  claim 74 , wherein the second region is a random N-mer sequence. 
     
     
         76 . The method of  claim 74 , wherein the second region is a targeted N-mer sequence. 
     
     
         77 . The method of  claim 74 , wherein the second region is a polythymine (poly-T) sequence. 
     
     
         78 . The method of  claim 62 , wherein the second nucleic acid molecule is coupled to a bead. 
     
     
         79 . The method of  claim 78 , wherein the second nucleic acid molecule is releasable from the bead upon application of a stimulus. 
     
     
         80 . The method of  claim 62 , wherein (b) comprises using a ligation reaction to generate the third nucleic acid molecule. 
     
     
         81 . The method of  claim 62 , wherein (b) comprises using an extension reaction to generate the third nucleic acid molecule.